1. Field of the Invention
Tissue plasminogen activator (tPA) is a serine protease involved with the dissolution of blood clots. The molecule has a number of distinct regions which display amino acid sequence homology with other naturally occurring polypeptides. Beginning at the N-terminus is a region homologous to fibronectin. The next region has homology with various growth factors. The growth factor region is followed by two kringle regions. Finally, there is the active site finding analogy with other serine proteases.
There are at least four different properties associated with tissue plasminogen activator and its ability to lyse blood clots in vivo. The first is the specific activity of the protease function in cleaving plasminogen to produce plasmin which in turn degrades fibrin. The second property is the sensitivity to inhibition by plasminogen activator inhibitor. The third property is the fibrin dependence of plasminogen activator for its plasminogenolytic activity. The fourth is the binding of tPA to fibrin surfaces. Each of these factors plays a role in the rapidity and the specificity with which plasminogen activator will cleave plasminogen to plasmin in the presence of blood clots. It would therefore be of substantial interest to be able to produce polypeptides having tissue plasminogen activator activity which will provide for improved properties in one or more of these categories.
2. Brief Description of the Relevant Literature
The use of mutagenesis to enhance native properties of a naturally occurring protein has been reported by Craik et al., Science (1985) 228:291; Rosenberg et al., Nature (1984) 312:77-80 and Wilkinson et al., Nature (1984) 307:187-188. Andreasen et al., EMBO J. (1984) 3:51-56, report that clipping at the cleavage site of tPA is necessary for protease activity. Ny et al., Proc. Natl. Acad. Sci. USA (1984) 81:5355, report the structure of human tPA and the correlation of introns and exons to functional and structural domains. A. J. van Zonneveld et al., Abstract from ISTH Meeting, Jul. 14-19, 1985, report on the relationship between structure and function of human tissue-type plasminogen activator. J. Biol Chem. (1986) 261:14214-14218. Opdenakker et al., EMBO Workshop on Plasminogen Activation (Amalfi, Italy, Oct. 14-18, 1985) report the effect of removal of carbohydrate side chains on tPA activity. Loskutoff et al., Proc. Natl. Acad. Sci. USA (1983) 80:2956-2960, and Loskutoff, Thrombos. Haemostos, THHADQ, 54(1):118 (S699) describe the properties of plasminogen activator-inhibitor.